Abstract Tendinopathy is a progressive degenerative disease that accounts for 20-30% of all musculoskeletal disorders and results in impaired tendon function and persistent pain. A primary cause of tendon degeneration is overuse (i.e., fatigue loading), which produces repeated microscale mechanical damage leading to the breakdown of load-bearing collagen fibrils. Furthermore, tendon degeneration is characterized by the accumulation of atypical tissue components (e.g., cartilaginous, fat, and calcium deposits), which additionally requires the synthetic activity of cells with abnormal (i.e., non-tenogenic) phenotypes. Endogenous tendon stem cells (TSCs) have the capacity to differentiate into multiple cell types and are hypothesized to undergo non-tenogenic differentiation in response to fatigue loading. Indeed, elevated or prolonged in vitro stretching of isolated TSCs has been shown to promote non-tenogenic differentiation. However, it is unknown how TSC fate is regulated by the specific changes in the native tendon microenvironment observed with fatigue loading. First, the actual in situ strains that cells experience in fatigue-damaged tissue have not been measured. Second, prior studies have shown that mechanical stretch can activate all non-tenogenic pathways suggesting that additional biophysical inputs (e.g., tissue stiffness and organization) are required to direct TSC commitment to a specific lineage. Finally, the intracellular mechanotransduction mechanisms that modulate TSC differentiation in response to changes in their mechanical microenvironment are unknown. Identifying how mechanical stimuli alter TSC fate and lead to tendon degeneration will elucidate the underlying cause of tendinopathy and will inform the discovery of novel treatments to prevent or reverse the degenerative process. The objective of this proposal is to determine how non-tenogenic TSC differentiation is regulated by fatigue-induced changes in the tendon mechanical microenvironment and to identify the mechanotransduction mechanisms that mediate this response. Specifically, this work aims to 1) determine how in situ microscale tendon mechanics are altered with fatigue loading, 2) isolate the unique effects of aberrant mechanical stimuli on TSC differentiation, and 3) identify the key mechanotransduction mechanisms that mediate TSC differentiation. This will be accomplished by measuring the local strains, stiffness, and organization of the cellular microenvironment in fatigue-damaged tendon using an ex vivo tissue culture model. To identify how non-tenogenic TSC differentiation is mediated by altered mechanical stimuli separate from other influences within the TSC niche (e.g., soluble factors), we will stretch isolated TSCs on substrates with different stiffness and topographies that match the measured in situ biophysical inputs. Finally, we will use various inhibitors of cytoskeletal tension and intracellular signaling to investigate the mechanotransduction mechanisms that convert the altered mechanical stimuli to non-tenogenic TSC differentiation. The findings of this work will identify the mechanisms by which tendon overuse induces non- tenogenic TSC differentiation and leads to tendon degeneration. Furthermore, the ex vivo tendon fatigue model provides a platform to evaluate novel treatments aimed at preventing degeneration and restoring tissue properties. Finally, this research will provide the applicant with the necessary training to become a successful independent investigator.